Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India

Background Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine–pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. Methods Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. Results A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. Conclusion The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.


Background
A global commitment and effort is underway to eliminate malaria, which is still a significant public health problem in several countries.In 2021, around 241 million malaria cases and 627,000 malaria deaths were observed globally [1].India contributes around 83% of cases in the South East Asia Region (SEAR), of which 61% were Plasmodium falciparum and 39% Plasmodium vivax infections [1,2].Universal access to malaria diagnostics and treatment is part of the World Health Organization (WHO) strategic framework for countries progressing towards elimination.Over the last several decades, malaria elimination strategies deployed by many national programmes include case management using robust surveillance, artemisinin-based combination therapy (ACT) and vector control strategies using long-lasting insecticidal nets (LLINs) and indoor residual sprays (IRS).These efforts are constantly challenged by the emergence of anti-malarial drug resistance of parasites and insecticide resistance of vectors.Plasmodium falciparum, which causes severe forms of the disease, is more prone to develop resistance against the anti-malarials, as compared to other human infecting malaria parasites [3][4][5].
Historically, uncomplicated P. falciparum malaria was treated with chloroquine (CQ).After the development of resistance to CQ, sulfadoxine-pyrimethamine (SP) was deployed as the frontline anti-malarial drug.However, parasites resistant to SP emerged rapidly, leading to the change in drug policy to use ACT for the treatment of uncomplicated malaria.Artesunate + sulfadoxine-pyrimethamine (AS + SP), a combination of ACT, has been recommended by the Indian National Drug Policy for Malaria as frontline therapy for the treatment of uncomplicated P. falciparum, except in northeastern states of India, where artemether-lumefantrine (AL) was introduced [6] .
Furthermore, most anti-malarials metabolise through ABC transporter and SNPs at codons N86Y and Y184F of Pfmdr1 confer resistance against partner drugs, such as mefloquine and lumefantrine thus limiting the therapeutic activity of artemisinin-based combinations [16].Since these mutations are linked to anti-malarial resistance, tracking them in the natural parasite population is an effective way to monitor for potential problems with anti-malarial drug efficacy, even before there are clinical reports [17].Baseline information about polymorphisms in marker genes associated with anti-malarial drug resistance will be helpful to predict the emergence of resistance among the parasite population in a given geographical region.
ACT was implemented starting in 2010 in Mandla district for the treatment of uncomplicated P. falciparum malaria.Therefore, the present study was conducted to assess the point mutations in marker genes Pfdhfr, Pfdhps, Pfk13, and Pfmdr1 associated with anti-malarial drug resistance among P. falciparum samples collected from Mandla, Madhya Pradesh, as a part of the Malaria Elimination Demonstration Project (MEDP).

Study area, population, and sample collection
This study was part of the Malaria Elimination Demonstration Project, which was a first-of-its-kind public-private-partnership between the Indian Council of Medical Research (ICMR) through the National Institute for Research in Tribal Health (NIRTH) Jabalpur, Government of Madhya Pradesh (GoMP), and the Foundation for Disease Elimination and Control of India (established by Sun Pharmaceutical Industries Ltd. as a not-for-profit entity) [18].
This study was carried out in Mandla district, Madhya Pradesh (between geo-coordinates 22° 02′ and 23° 22′ N and 80° 18′ and 81° 50′ E), which is mainly inhabited by Scheduled Tribes (59%) [19].The district has an area of 8771 km 2 divided into nine development blocks (Fig. 1) and 1233 villages; the total population is 1,140,765 population [18].Dried blood spots were collected from finger pricks as described previously during the active door-todoor fever survey [20] and Mass Screening and Treatment (MSaT) strategies [21] during 2019-2020.The detailed methodology of sample collections and results of malaria diagnosis using rapid diagnostic tests, microscopic smear examinations, and Polymerase Chain Reaction (PCR) findings were reported elsewhere [20][21][22].

Genomic DNA extraction and parasite genotyping
Molecular analysis was done from 393 P. falciparumpositive dried blood spot samples during 2019-2020.Genomic DNA was isolated from the dried blood spots using the Chelex method, as described earlier [21,22].The presence of Plasmodium species was determined using species-specific nested PCR by targeting 18Sr RNA gene, a technique reported elsewhere [21,22].

Nucleotide sequencing
The PCR amplicons were purified using exonuclease I and shrimp alkaline phosphatase, following the manufacturer's instructions.The purified product was used with the ABI Big dye Terminator Ready Reaction Kit Version 3.1 for DNA sequencing.The sequencing PCR was performed in a volume of 10 µL with 0.5 µL of Terminator Ready Reaction Mix (TRR), 3.2 pmol of gene specific forward and reverse primer (both directions) and sequencing buffer.Cycling conditions for the sequencing PCR include 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s and extension at 60 °C for 4 min.Sequencing was performed on a 3130XL genetic analyser (Applied Biosystems, USA).Sequencing results were analysed by V5.4 software (Applied Biosystems, USA), and contigs were prepared using Bioedit sequence alignment editor version 7.2.3.

Statistical analysis
Data was entered in Microsoft Excel 2013 Worksheet.Qualitative (Categorical) variables were coded numerically, and frequency with percentage distribution was tabulated.Pearson's Chi-square or Fisher's exact test was applied for appropriate statistical comparison of independent proportions.Bivariable logistic regression analysis was performed to estimate the association of independent factors such as endemicity, age group, symptoms, and year with Pfdhfr, Pfdhps, Pfk13, and Pfmdr1 gene mutations.All the statistical analysis has been performed using R version 4.2.2 for Windows (R Foundation for Statistical Computing, Vienna, Austria).

Results
A total of 335 out of 17,405 during the active fever survey and 221 out of 24,357 samples during the MSaT were P. falciparum positive by PCR.A total of 393 out of 556 P. falciparum positive samples were available in sufficient quantity and used for molecular genotyping of Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1 genes using the Sanger sequencing method and good read from both the direction were used for the sequence analysis.Mutations were identified with using 3D7 as reference strain.

Analysis of pfdhfr and pfdhps mutations
A total 199 P. falciparum samples were successfully analysed for both Pfdhfr and Pfdhps genes.Out of the five Pfdhfr mutations (A16V, N51I, C59R, S108N/T, and I164l) conferring pyrimethamine resistance, only three mutations at codons N51I, C59R, and S108N were found as single, double or triple mutations (Table 2).In addition to the above five functional mutations, we have found a novel mutation at L46S in the triple mutant combination.Overall, 58% of parasite isolates were found to harbour mutant genotypes, while only 42% had wild-type (sensitive) genotypes.Majority of them were double mutants (53.3%) at codon C59R and S108N followed by triple mutants (3.5%) and 1.0% single mutants (Fig. 2).Four mutant haplotypes were determined in the Pfdhfr gene.More (62%, 84/135) samples were found with resistant mutants in 2020 compared to 2019 (48%, 31/64).No significant difference in the prevalence of mutant genotype was observed among the symptomatic vs. asymptomatic cases, adult vs. children, and in different malaria endemic areas (Table 3).
When the data of mutations of the two genes (Pfdhfr and Pfdhps) was analysed, we found that only 1% was wild type.A quadruple mutant of Pfdhfr (N51I, C59R, S108N) and Pfdhps (S436A) was found in only four cases.However, the triple mutant Pfdhfr (C59R, S108N) and Pfdhps (G437A) were found in 78/106 (73.6%) samples (Table 2).The study has not found any resistant associated mutations for Pfdhfr at codon 164 and for Pfdhps at codons 581 or 613.The six samples with triple mutant   Pfdhfr N51I, C59R, S108N were distributed throughout the district (two cases from low endemic areas, three from moderate and one from high) in 2020.Similarly, four pfdhps mutations at codon S436A were distributed throughout the districts (one case from low endemic areas, two from moderate and one from high) in 2020.
Overall, there was no significant difference in the distribution of Pfdhfr and Pfdhps mutants observed over the 2-year study period (Fig. 1).

Analysis of Pfmdr1 mutations
The Pfmdr1 gene was analysed from 141 samples, and mutation at N86Y was found in 13.5% samples and did not find any mutations at codon Y184F (Fig. 2).The study revealed that 51% of samples had synonymous mutations at different codons (182, 183, 215, 231 and 271).
The study has also analysed various independent factors associated with antimalarial drug resistance markers, such as malaria endemicity, age group, symptoms, and year of sample collection.The analysis revealed a significantly higher number of mutations in Pfmdr1 in high endemic areas, Pfk13 in the asymptomatic group, and Pfdhps in children under 14 years of age (Table 3).

Discussion
Since 2010, ACT has been used in India to treat uncomplicated P. falciparum malaria after significant resistance was found against CQ and SP [12,[23][24][25][26][27][28][29][30][31][32][33].Due to the emergence and spread of anti-malarial drug-resistant parasites, monitoring of molecular markers has become an essential component of malaria control strategies.The present study investigated the mutations in the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes of P. falciparum samples collected as part of MEDP.
Mutations in Pfdhfr and Pfdhps genes associated with SP resistance have been reported in India and other countries, and parasites harbouring quintuple mutations of Pfdhps (437G and 540E) and Pfdhfr (51I, 59R, and 108N) genes are known to be resistant to SP [34,35].It has been documented that single point mutation in these two genes signals early signs of the improper action of the drugs, while the double mutations may indicate a decreased parasite sensitivity to the drug, and multiple mutations (triple or more) raise concerns for drug failures [36].In the present study, the triple mutations in the Pfdhfr (C59R, S108N) and Pfdhps (G437A) genes were found in 73.6% of samples, which indicates circulation of possible resistant forms of P. falciparum parasites in the study area.Similar triple mutation-bearing parasites have been reported in central India [9,26,27,[37][38][39].However, studies conducted in the eastern (Odisha, West Bengal) and North Eastern states have revealed high levels of (quadruple and quintuple) mutations that are associated with drug failures [11,24,26,[40][41][42][43].
In the present study, only four samples out of 235 were found to have quadruple mutants of Pfdhfr (N51I, C59R, S108N) and Pfdhps (S436A).Molecular surveillance should be continued in the study area to monitor the emergence and spread of SP resistance as low level of quadruple mutants have already been observed.
Mutation in Pfmdr1 N86Y is known to contribute to multi-drug resistance.In this study, 13.5% of samples were found to bear N86Y mutation, and 51% had synonymous mutations.The study has also revealed that synonymous mutations were neither at codon 86 nor at 184, which are crucial for conferring drug resistance.Studies have shown that the prevalence of N86Y mutation varies across the country and the South East Asia region depending upon the drug pressure and transmission intensity [16,28,32,33,39,53].It has been shown that N86Y mutation has a positive modulation effect when present in combination with other drugresistant mutations.These observations indicate that the dynamic nature of evolving parasite populations exposed to different antimalarial drugs may influence the emergence of drug resistance.The triple mutant Pfdhfr N51I, C59R, S108N were distributed throughout the district in 2020.This could be due to several reasons for the higher number of mutations in the year 2020 as compared to 2019.MEDP started the active fever cases screening and treatment in the month of September 2017 and continued till 2021 to eliminate the indigenous malaria cases from the study area.The project also carried out MSaT strategies to resolve the malaria hotspots and asymptomatic cases during 2018 and 2019, which could have put drug pressure on the parasite resulting in the emergence of mutationsThe present study provides the status of molecular markers associated with the P. falciparum drug resistance.However, the therapeutic efficacy and in-vitro assessment were not part of the study.Such studies should be carried out in future if there are reports of delays/failures of ACT.

Conclusion
The study found no mutations linked to ART resistance in the Pfk13.However, the finding of triple mutations in Pfdhfr and Pfdhps could present a problem for the use of AS + SP for the treatment of uncomplicated P. falciparum malaria in the study areas.Therefore, there is a need for continued molecular surveillance to quantify the presence of mutation-bearing parasites.Molecular surveillance, together with in-vivo efficacy and therapeutic efficacy studies using appropriate epidemiologic designs, would guide policymakers to make appropriate decisions for making any changes in the treatment regimen.
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Fig. 1
Fig. 1 Map of India showing Mandla district in Madhya Pradesh, the study site divided in nine different block with different level of mutation in anti-malarial resistance markers

Table 2
Different haplotype of Pfdhfr gene and their association with Pfdhps haplotypes (NIL: dhps gene didn't successfully analysed and WILD TYPE: no mutations in the analysed samples)

Table 3
Logistic regression analysis of the various factors associated with the different anti-malarial markers from the study sites